The overall objective is directed toward gaining further insight into intermediary nitrogen metabolism through the study of nitrogen transferring reactions, particularly those involved in arginine synthesis and urea formation from citrulline which are catalyzed by argininosuccinate synthetase and argininosuccinase. The several objectives of the proposed projects center on problems relating to active site studies of argininosuccinate synthetase and argininosuccinase, and on enzymological problems of genetic and biological significance. Completion of active site investigations on argininosuccinase will pinpoint the proton donor in the beta elimination reaction mechanism and will contribute to primary structure in the active site region for its bearing on substrate binding and structure-function relationships. Completion of studies on affinity of analogues will establish structural requirements for activity as a substrate for this enzyme and may disclose inhibitors potentially useful for selective inhibition in the multienzyme sequence of the urea cycle enzymes. With regard to argininosuccinate synthetase, active site studies with reagents known to be reactive at the binding site of the pyrophosphate grouping of ATP can provide insight into the nature of the ATP-binding region of the active site and of the effect of aspartate on pyrophosphate binding. A group of closely related studies are directed (a) to the completion of a comparative study on bovine brain argininosuccinase which is of genetic interest and has bearing also on problems of tissue differentiation, brain development and metabolic function, and (b) completion of very sensitive radioassays applicable to tissue homogenates prepared from hepatic and extrahepatic sources.